In both research applications and clinical diagnosis, it is considered desirable to link various monomeric units to form oligomeric structures. Examples of such structures include oligonucleotides, oligopeptides and the like.
For example, a known technique for determining the presence of a target nucleotide sequence in either RNA or DNA is to perform a nucleic acid hybridization assay. In such an assay, a nucleotide probe, typically an oligonucleotide, is selected having a nucleotide sequence complementary to at least a portion of the target nucleotide sequence. Typically, the probe is labelled to provide a means whereby the presence of the probe can be readily detected.
When the labelled probe is exposed to a sample suspected of containing the target nucleotide sequence, under hybridizing conditions, the target sequence will hybridize with such a labelled probe. The presence of the target sequence in the sample can then be determined qualitatively or quantitatively, usually after separating hybridized and non-hybridized probes and determining the presence and/or amount of the labelled probe which hybridized to the test sample.
Prior methods for linking a label to a nucleotide probe have generally utilized a single label attached to a nucleosidic monomeric unit, and then incorporated one or more of the nucleosidic monomeric units into the probe. For example, analogs of dUTP and UTP containing a biotin moiety have been chemically synthesized and incorporated into polynucleotides 24!. Such biotin-labelled nucleotides may then be incorporated into nucleic acid probes of biological or synthetic origin.
Other methods for labelling nucleotide probes have been proposed which allow labels to be randomly linked to nucleotides in a nucleotide oligomer. Numerous proposals have been made for incorporating multiple modified nucleosides or non-nucleosidic monomeric units into oligonucleotides with a view towards enhancing the detectability of the labelled probe and the target nucleotide sequence. In addition, it has been considered desirable to provide a means for attaching multiple labels to a single monomeric unit in an oligonucleotide probe.
However, it has been demonstrated that use of many such labelled nucleotides in a probe can reduce the stability of the hybrid formed with a target nucleotide sequence, particularly when multiple labels are present. Such reduced hybrid stability has been demonstrated for nucleic acid probes of biological origin possessing multiple biotin moieties, for synthetic oligonucleotides possessing multiple fluorescein labels, as well as for synthetic oligonucleotides possessing biotin and fluorescein labels.
In addition, derivatives of nucleoside linking phosphate groups have been disclosed, the nucleophilic moiety of which can be labelled following their incorporation into an oligonucleotide. However, such compounds, being based on nucleoside derivatives, would be expected to exhibit some of the disadvantages discussed above for nucleoside-based derivatives.
More recently, 2-amino-1,3-propanediol structures have been used to label oligonucleotides with reporter groups 6!.
A number of methods to incorporate multiple reporter molecules into oligonucleotides have been described 1-8!. These utilize linear addition of labeled phosphoramidites or analogs. Since only one label is added per synthesis cycle, the number of labels that can be incorporated is limited.
Other methods for the introduction of multiple amino groups involve the use of polylysine--oligonucleotide conjugates 9-10!. These methods require the use of a combination of solid-phase peptide and oligonucleotide chemistries, a considerable disadvantage.
As one means of introducing multiple labels, as well as providing other beneficial characteristics, the production of "branched" nucleotide oligomers has been proposed. Phosphoramidites that introduce "branched" structures having two 5' ends into the nascent oligomer allow the addition of labels exponentially. For "n" synthesis cycles, the number of labels added is 2.sup.n. Several non-nucleosidic branching phosphoramidites are known 11-18!. These non-nucleosidic phosphoramidites are based on linear, acyclic alkanetriols.
A nucleosidic branching phosphoramidite based on a modified deoxycytidine derivative has been successfully used in nucleic acid hybridization assays 11, 19-22!.
Many of the phosphoramidites used for "branching" have the inherent disadvantage that they introduce additional centers of chirality into the final structure. This disadvantage is overcome by the use of an achiral non-nucleosidic phosphoramidite 23!. In this case, phosphoramidite synthesis is a multi-step process and its use requires modification of standard DNA synthesis protocols.
Thus it is considered desirable to provide multifunctional reagents which demonstrate high coupling efficiency and thus provide higher yields of labelled oligomer.
Furthermore, it is also considered desirable to provide a class of such reagents for use in forming nucleotide oligomers which permit the resultant oligomers to hybridize with efficiencies approaching those of oligomers which contain only native nucleosidic monomeric units.
It is also considered desirable to provide such a reagent which is also capable of use in non-nucleotidic oligomers, such as in oligopeptide oligomers.
It is further considered desirable to provide such reagents which permit the use of standard synthetic chemistries.